Phenotypic Bacterial Isolates, Antimicrobial Susceptibility pattern and Associated factors among Septicemia Suspected Patients at a hospital, in Northwest Ethiopia: Prospective cross-sectional study

Background Septicemia is potentially fatal infection caused by pathogenic bacteria infiltrating the bloodstream, resulting in morbidity and mortality among Ethiopian hospital patients. Multidrug resistance is a therapeutic challenge in this patient population. There is an insufficiency data among hospitals in Ethiopia. Hence, this study aimed to assess the phenotypic bacterial isolates, antimicrobial susceptibility pattern, and associated factors among septicemia suspected patients. Methods Prospective cross-sectional study was conducted among 214 septicemia suspected patients from February to June 2021 at Debre Markos Comprehensive Specialized hospital in northwest, Ethiopia. Blood samples were collected aseptically and processed to identify bacterial isolates by using different standard microbiological procedures. Antimicrobial susceptibility pattern was performed using the modified Kirby Bauer disc diffusion on Mueller Hinton agar. Epi-data V4.2 was used to enter data and SPSS V25 for analysis. The variables were assessed using a bivariate logistic regression model with a 95% confidence interval, and declared statistically significant; P-value was < 0.05. Results The overall bacterial isolates was found 45/214 (21%) in this study. Gram-negative and positive bacteria were 25/45(55.6%), 20/45(44.4%) respectively. The most common bacterial isolates were Staphylococcus aureus12/45 (26.7%), Klebsiella pneumoniae 8/45(17.8%), Escherichia coli 6/45 (13.3%). Gram-negative bacteria showed susceptibility to amikacin (88%), meropenem, imipenem (76%) but, (92%) resistance to ampicillin, (85.7%) amoxicillin-clavulanic acid. S.aureus (91.7%) was resistance to Penicillin, (58.3%) cefoxitin and (75%) susceptible to ciprofloxacillin. S.pyogenes and S.agalactia were (100%) susceptible to Vancomacin. Multidrug resistance was found in 27/45(60%) of the bacterial isolates. The main predictors related to patients suspected of septicemia were prolonged hospitalization (AOR = 2.29, 95% CI: 1.18, 7.22), fever (AOR = 0.39, 95%CI: 0.18, 0.85) and length of hospital stay (AOR = 0.13, 95%CI: 0.02, 0.82). Conclusions Incidence of bacterial isolates among septicemia suspected patients were high. The majority of the bacterial isolates were multidrug-resistant. To prevent antimicrobial resistance, specific antibiotic utilization strategy should be applied.


Background
Septicemia is a life-threatening bloodstream infection that is a major public health concern around the world if not treated with appropriate antimicrobials [1]. Septicemia is when the bacteria enter the bloodstream, and cause blood poisoning which triggers sepsis and comparable with bacteremia with sepsis, and systemic inflammatory response syndrome (SIRS) [2,3]. It causes illness and deaths in all group of population [4], especially in immunocompromised patients, an intensive care unit (ICU), elders, and children [5], cancer patients [6,7], and patients living with human immunodeficiency virus (HIV) [8]. The incidence rate of septicemia is increased due to previous hospitalization, length of hospital stay, unexplained fever (> 37 o c), age groups, serious injury, chronic antibacterial therapy, comorbidities, invasive medical procedures, respiratory disease, urinary tract disease, gastrointestinal disease, HIV/AIDS, hematological disorders are the factors that increase the severity of septicemia [4,6,9,10]. Different Gram-negative and Gram-positive bacteria are common, and vary depending on geographical location and time [11][12][13][14].
According to recent scientific publications, the global burden of bacterial infection of the blood was 48.9 million new cases of septicemia in 2017, with 11.0 million septicemia-related deaths, accounting for 20% of all deaths worldwide [15]. The World Health Organization (WHO) estimates that 41% of all global sepsis cases in 2020 were children under the age of five, with a death rate of 42% among intensive care patients with septicemia [16,17]. One in two cases of sepsis in intensive care units (ICUs) results from healthcare-associated (HCA) infections. Mortality in HCA septicemia from hospitalized adult patients ranges from 20 to 30%, and in pediatric ICUs from 20 to 50% of deaths worldwide among 200,000 cases reported [16][17][18][19][20]. On the other hand, 40% of infants diagnosed with septicemia were died, with the majority of deaths occurring in developing countries, making it difficult to control worldwide [21,22]. More than 19 million septicemia cases and 5 million septicemia-related deaths are estimated to occur each year in low and middle-income countries including Ethiopia [15,23].
Antimicrobial management is initiated empirically in almost all septicemia-related cases prior to the results of blood culture [22]. As a result, patients who are improperly treated may have a 100% mortality rate [24] and the emergence of antimicrobial resistance in all types of pathogenic bacteria has worsened in resource-constrained countries [25,26]. Furthermore, self-prescription, misuse, and over-prescription of antimicrobials in the absence of true etiological agent identification pose a significant challenge to antimicrobial resistance control in Ethiopia. As a result, there is little information available about commonly prescribed antibiotics and their susceptibility profiles, as well as the epidemiology of bacterial isolates from suspected septicemia patients. Thus, this study aimed to assess the phenotypic bacterial isolates, antimicrobial susceptibility pattern, and associated factors among septicemia suspected patients at a hospital, in Northwest Ethiopia.

Study design, period and settings
A hospital-based prospective cross-sectional study was conducted in septicemia suspect patients referred and admitted to Debre Markos Comprehensive Specialized Hospital (DMCSH) from February to June 2021. The hospital is located in Amhara National Regional state's East Gojjam zone, at Debre Markos town. DMCSH is a 218-bed hospital and the largest public comprehensive hospital in the zone, receiving patients directly from the community as well as referrals from district primary hospitals and health centers. DMCSH provides health services to over 5 million people in catchment area, and neighboring border neighborhoods. It has various wards of ICU, medical, pediatric, surgery, emergency, gynecology, and medical laboratory services.

Inclusion criteria
During the study period, the study populations were included when all patients have over the age of one month who was suspected of having septicemia and given consent and assent.

Exclusion criteria
Patients who were seriously ill and did not provide a blood sample during the data collection period, contaminated blood samples, were excluded from the study.

Sample size determination and sampling
The sample size was calculated with the formula (n= (Z 2 ) 2 P (1-P) /d 2 ). By reviewing similar previous studies, the maximum sample size was obtained from a study conducted in southern Ethiopia, with a prevalence/proportion of septicemia was 15.8% (0.158) [1]. As a result, Conclusions Incidence of bacterial isolates among septicemia suspected patients were high. The majority of the bacterial isolates were multidrug-resistant. To prevent antimicrobial resistance, specific antibiotic utilization strategy should be applied. Keywords Bacterial isolates, Septicemia, Antimicrobials susceptibility, Ethiopia

Operational definition
Multidrug resistance: Multidrug resistance is defined in this study as simultaneous resistance to three or more antimicrobial agents from different antimicrobial groups [27]. Septicemia: when the patients classified as having septicemia the physician suspected two or more symptoms of SIRS, temperature > 38°c or < 36°c heart rate > 90/ min, respiratory rate > 20/min and white blood cell count > 12,000/mm 3 or < 4000/mm 3 to the infection [2]. Fever: The patients have greater than > 37°c during the study period (yes or no question).

Data collection, specimen processing and culturing for identification of bacterial isolates
To collect socio-demographic, laboratory, and clinical data, semi-structured questionnaires were prepared from different previous literatures [4,9,10,12,13,28]. Faceto-face interviews with trained nurses and laboratory personnel were used. The blood samples were collected by laboratory technicians and experienced nurses who had undergone training. The vein puncture site was properly disinfected with 70% alcohol and 2% iodine tincture. Within a 30-minute interval, two bottles of blood (5 mL of blood for patients over the age of five and 1-3 mL of blood for patients under the age of five) were collected aseptically from both hands for each patient [29]. After obtaining the blood, it was inoculated at bedside into Tryptone Soy Broth (TSB) (Oxoid, Hampshire, UK) maintaining a minimum of blood to broth ratio 1:10. The TSBs were prepared according to the blood specimen collected; 10ml, 20ml, 30ml, and 45ml. Medical microbiology specialists were performed blood culture processing, biochemical testing, and antimicrobial susceptibility pattern of the isolate. Gram-positive bacteria were tested using catalase, coagulase, the CAMP test, and the Bacitracin test.

Antimicrobial susceptibility testing
All blood culture isolates were tested for antimicrobial susceptibility using modified Kirby Bauer disc diffusion methods on Mueller-Hinton agar (MHA) plates (Oxoid, Hampshire, UK) using clinical and Laboratory standards Institute (CLSI ) 2021 guideline [30]. Fresh 2 to 5 pure colonies from BAP or CAP were selected and transferred to 5 ml of sterile nutrient broth (Oxoid, Hampshire, UK.) and mixed to make the suspension homogeneous before being transferred into sterile normal saline with a sterile pipette. Finally, turbidity was visually adjusted with sterile normal saline to match a 0.5 McFarland standard. Using a sterile swab, the suspension was then inoculated uniformly over the entire surface of an MHA plate. For 3-15 min, the inoculated plates were left at room temperature to dry. The antibiotic discs were placed on MHA using sterile forceps at a distance of 24 mm between each disk and 15 mm from the border. The antimicrobial discs were used in the concentrations indicated in brackets, were chosen based on availability, and were frequently prescribed for the treatment of bacterial infections in hospitals throughout Ethiopia. Antibiotics used for this study were: Ampicillin (AMP) (

Quality control
Questionnaires had been pre-tested on 5% questionnaires from fenote Selam General Hospital to ensure consistency. The questionnaires were written in English, translated into Amharic, and then back translated into English. A half-day training session was provided for laboratory personnel and nurses. Standard operating procedures (SOPs) were strictly followed throughout the laboratory analysis in this study to ensure the quality of the process from pre-analytical to post-analytical.
To ensure the sterility of the media, 5% of all prepared culture media were incubated overnight at 37

Data analysis and interpretation
The Epi-Data V.4.2 computer program was used to enter and clean the data before exporting it to the SPSS V.25 software package for analysis. Cross-tabulation of each variable was used to ensure the data's completeness and consistency. Descriptive analysis was used like tables, graphs. A binary logistic regression model was used to demonstrate any relationship between independent and outcome variables. In the bivariate analysis, a variable with a p-value of ≤ 0.25 was entered into the multivariate analysis to identify variables that were independently associated with the outcome variable. The presence and strength of association between independent and outcome variables were calculated using an adjusted odds ratio with a 95% confidence interval (CI). The Hosmer-Lemeshow test was used to assess the model's fitness test [31]. A p-value of < 0.05 was regarded as an indicator of statistically significant results.

Socio-demographic and clinical characteristics
This study was included 214 septicemia suspected patients. Of these, 109 (50.9%) were males. The median age of the participants with their interquartile range was 14 ± 9.3 years, (64%) had fever, and 43 (20.1%) had previous medical procedures ( Table 1).

Factors associated with blood culture positivity and septicemia
In the bivariate analysis, socio-demographic and clinical variables were revealed to be associated with septicemia in the bivariate. Patients with a temperature greater than 37 0 c and a previous medical procedure had a risk factor, but it was not statistically significant at the 95% CI. In this study, those who had a history of antibiotic use were 2.21 times more likely to have a positive blood culture than those who had never used antibiotics before, and this was statistically significant at (95% CI;1.11-4.39, p = 0.024). The candidate variables for multivariate analysis were age, ward type, fever, previous hospital admission, admission date in the hospital, previous medical procedure, antibiotic usage, and temperature. A multiple logistic regression method using the backward variable selection method revealed that only three variables were associated with blood culture positivity. According to the multiple logistic regression analysis, patients who had previously been admitted to the hospital were 2.9 times more likely to develop septicemia than patients who had never been admitted to the hospital. In this study, patients with fever (temperature > 37 o C) were 61% less likely to develop septicemia than their counterparts (Table 2).

Antimicrobial susceptibility patterns of bacterial isolates The susceptibility of Gram-negatives
Gram-negative bacteria (n = 25) were isolated from blood cultures of patients suspected of having septicemia and tested for sensitivity to 12 antimicrobial discs (Table 3).

Multidrug resistance (MDR)
The prevalence of multidrug resistance in our study was 27/45 (60%). Whereas three (6.7%) blood bacterial isolates were sensitive to all antimicrobials tested, one isolated bacteria was resistant to all antibiotic groups tested. However, when drug resistance patterns were compared to species-specific observed, two (100%) of k.rhinose, two (100%) of Serratia spp, six (75%) of K.pneumoniae and three (75%) of P.aeruginosa were MDR pathogens in this study ( Table 5).

Discussions
The findings of this study revealed that the overall prevalence of bacteria isolated from septicemia suspected patients via the blood culture method was 21%, which was consistent with studies conducted in a variety of countries around the world, including Ethiopia, India,  [43], Arba Minich (11.3%) [28], Nepal (10.6%) [14], Kuwait (2.3%) [19], New Zealand (9.5%) [44] India (4.4%) [45], Bangladesh (13.2%) [45], Ghana (11.2%) [46], and Southern Africa (5.5%) [47]. The possible reason for this variation might be explained by the fact that difference in the geographical location, the  Table 3 Antimicrobial susceptibility pattern of Gram-negative bacteria isolated from blood culture for patients suspected of septicemia at a hospital, in Northwest Ethiopia, 2021 study population, method of study design, sample size, epidemiological variation, implementation of infection prevention and control, blood culture diagnostic system, study duration, and health care policy systems in these different countries.
In the present study, Gram-negative bacteria were more predominantly isolated than Gram-positive bacteria (55.6%) and (44.4%), respectively. Similarly, concordant findings were observed in India (51.2%) [37] Iran (55.4%) [35] Nepal (52.3%, 50.5%) [14,48]. However, our finding was lower than other studies conducted, where Grampositive bacteria was the predominant isolated bacteria than Gram-negative bacteria in India (60.9%61.4%) [43], Jimma (53.3%) [1], Addis Ababa (77.4%) [39], Gondar (66%) [11]. The varying percentages could be due to methodology difference and the patient safety practice, diagnostic method and sample size. In the current study the prevalence of S. aureus (26.7%), K.pneumoniae (17.8%), E. coli (13.3%), CONS (8.9%), and P.aeruginosa (8.9%) were the main isolate, this was in line with Indonesia K.pneumoniae (17%), P.aeruginosa (12%) [49]. Greater or a lesser amount of similar results have been seen in studies conducted in septicemia suspected patients from different regions in Ethiopia, Addis Ababa (50%, 26.21% 14.02%) [39], Mekelle (37.5%,11.1%) [12], and Southern Ethiopia (32.2%,14.3%) [28]. Due to these bacteria are   : resistance for only one antimicrobial groups, R2: resistance for two antimicrobial groups, R3: resistance for three antimicrobial groups, R4: resistance for four antimicrobial groups, R5: resistance for five antimicrobial groups, R6: resistance for six antimicrobial, and R7: resistance for seven antimicrobial groups normal microbiota of the skin, and the majority of the time found in the hospital areas as a result cause infection in immune susceptible patients. In the present study, different associated factors were evaluated for various associations with septicemia. Nevertheless, not all of the socio-demographic variables in the current study were statistically associated with septicemia. However, this study indicated that males were more infected than females (23.8% vs. 19.2%), respectively, but there was no a statistically significant difference in gender variation (p = 0.632). This slight variation has been reported in different studies [11,12,50]. Patients in under-five age group were more infected with septicemia as compared to the adult age group. The possible reason for this is the children have more susceptible to infection due to less immune system and colonized by normal microbiota. Nevertheless, in this study the category of age groups were not statistically significant (p = 0.186). This finding disagrees with the research done in southern Ethiopia [28], Rwanda [51] Nepal [14], and the USA [52]. The possible reason for this difference might be due to the none representative of samples in each ward from this study. Previous hospitalization in this study was 2.9 times more likely associated with septicemia in the current study. This research finding is consistent with the study conducted in Ethiopia AOR = 5.54 and AOR = 3.2 [8,28]. This association might be due to health care associated infections, high number of patients admitted to hospitals for prolonged periods, delayed request for blood culture, and a weak patient safety, and management system in the hospital. As a result, fast intervention had a must to minimize the infection.
The overall resistance of Gram-positive and Gramnegative bacteria in this study were 25-66.7% and 8-92% respectively, which was similar to the result reported in Mekelle [12], Jimma [1]. This condition gives severe anxiety to the population. This high rate of resistance might indicate misuse and inappropriate usage of the antibacterial drugs. However, the resistance is different from the study conducted in Gondar where the rate of resistance for Gram-positive bacteria ranged from (23.5-58.8%) and Gram-negative bacteria (20% 100%) [11]. The observed variation may be due to the arbitrary use of antimicrobials in the study area, self-prescribing antimicrobials in private drug stores and empirical treatment of hospital acquired infections without real etiological agent identification.
S.aureus was (91.7%) resistant to penicillin, but susceptible to (75%) gentamicin, and (83.3%) ciprofloxacillin. This was similar with reports conducted in different countries 40-97% [10,20,33]. This is due to the unrestrained use of antimicrobials without sensitivity testing in the study area. (66.7%) of MRSA was detected. In order of, the same report was done in Indian and Ethiopian researches [6,20,45]. The incident of MRSA is more common because of the haphazard use of higher antimicrobials empirically and genetic proficiency to obtain antibiotic resistance. S. pyogenes were 100% sensitive to cefepime, ampicilin, and vancomycin from our study. This was in line with the study reports, which had in 100%, 87% [13,33]. Vancomycin was 100% sensitive to S. agalactia. This finding was similar to the reports done in Mekelle [12]. The possible reason for the absence of vancomycin resistant bacteria in this study is that the use of vancomycin by clinicians is controlled in the management of patients in the study setting, and in the nation as an entire.
K.pneumoniae, E.coli, P.aeruginosa, K.rhinose, and Serratia spp in this study were (50-100%) sensitive to meropenem, imipenem, and amikacin. This finding was similar to the reports [20,28,37,45]. K.pneumoniae and E.aerogenes were 100% resistant to ampicillin, amoxicillin -clavulanic acid. In addition, E.coli (83.3%) for gentamicin and P.aeruginosa (75%) for gentamicin and ampicillin resistance were observed in this study. These were consistent to other studies conducted in Ghana 74% [46], Ethiopia 86% and 94% [8,41], India 93%, 87% [45,53]. The finding of this study indicates most of the gram-negative bacteria were 50-100% resistance to ciprofloxacillin, gentamicin, ceftriaxone, chloramphenicol, trimethoprinsulfamethoxazole, and ceftazidime. This was concordant with the research done in Cameroon 45-100% [24], and Ethiopia [8,41,43]. The limitation of this study was done in a single hospital, small sample size, short duration and only a phenotypic test was done, which may under estimate the prevalence of bacteria for the different geographical areas. This study has not detected fungemia and anaerobic bacteria due to scarcity of the media.

Conclusions
In this study, the overall prevalence of blood culture positivity among septicemia suspected patients was moderately high. Gram-negative bacteria were the predominant etiological agents for septicemia in the study populations. S.aureus, K.pneumoniae, E.coli, CONS, and P. aeruginosa were the predominant causative agent for septicemia. Conversely, S. aureus was the highest prevalence in the bacterial isolates. Previous hospital admission, fever, and length of hospital stay were significantly associated with septicemia in our study. Gram-negative bacteria were highly resistant to ampicillin, amoxicillin-clavulanic acid, ceftriaxone, chloramphenicol, and trimethoprim-sulfamethoxazole, but susceptible to meropenem, imipenem and amikacin. S. aureus was highly resistance to penicillin. While, susceptible to gentamicin and ciprofloxacillin. High multi-drug resistance rates were observed in most bacterial isolates. The study calls researchers to conduct a further study with a large sample size, with better study